Glycosylation of bacterial antigens changes epitope patterns.

Introduction: Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines. Methods: Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level. Results: The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination. Discussion: This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.

Prostate-Specific Membrane Antigen (PSMA)-Positive Extracellular Vesicles in Urine-A Potential Liquid Biopsy Strategy for Prostate Cancer Diagnosis?

All cells release extracellular vesicles (EVs) to communicate with adjacent and distant cells. Consequently, circulating EVs are found in all bodily fluids, providing information applicable for liquid biopsy in early cancer diagnosis. Studies observed an overexpression of the membrane-bound prostate-specific membrane antigen (PSMA) on prostate cancer cells. To investigate whether EVs derived from communicating prostate cells allow for reliable conclusions on prostate cancer development, we isolated PSMA-positive, as well as CD9-positive, EVs from cell-free urine with the use of magnetic beads. These populations of EVs were subsequently compared to CD9-positive EVs isolated from female urine in Western blotting, indicating the successful isolation of prostate-derived and ubiquitous EVs, respectively. Furthermore, we developed a device with an adapted protocol that enables an automated immunomagnetic enrichment of EVs of large sample volumes (up to 10 mL), while simultaneously reducing the overall bead loss and hands-on time. With an in-house spotted antibody microarray, we characterized PSMA as well as other EV surface markers of a prostate cohort of 44 urine samples in a more simplified way. In conclusion, the automated and specific enrichment of EVs from urine has a high potential for future diagnostic applications.

Potential and challenges of specifically isolating extracellular vesicles from heterogeneous populations.

Extracellular vesicles (EVs) have attracted interest due to their ability to provide diagnostic information from liquid biopsies. Cells constantly release vesicles divers in size, content and features depending on the biogenesis, origin and function. This heterogeneity adds a layer of complexity when attempting to isolate and characterize EVs resulting in various protocols. Their high abundance in all bodily fluids and their stable source of origin dependent biomarkers make EVs a powerful tool in biomarker discovery and diagnostics. However, applications are limited by the quality of samples definition. Here, we compared frequently used isolation techniques: ultracentrifugation, density gradient centrifugation, ultrafiltration and size exclusion chromatography. Then, we aimed for a tissue-specific isolation of prostate-derived EVs from cell culture supernatants with immunomagnetic beads. Quality and quantity of EVs were confirmed by nanoparticle tracking analysis, western blot and electron microscopy. Additionally, a spotted antibody microarray was developed to characterize EV sub-populations. Current analysis of 16 samples on one microarray for 6 different EV surface markers in triplicate could be easily extended allowing a faster and more economical method to characterize samples.

DjinniChip: evaluation of a novel molecular rapid diagnostic device for the detection of Chlamydia trachomatis in trachoma-endemic areas.

BACKGROUND: The clinical signs of active trachoma are often present in the absence of ocular Chlamydia trachomatis infection, particularly following mass drug administration. Treatment decisions following impact surveys and in post-control surveillance for communities are currently based on the prevalence of clinical signs, which may result in further unnecessary distribution of mass antibiotic treatment and the increased spread of macrolide resistance alleles in 'off-target' bacterial species. We therefore developed a simple, fast, low cost diagnostic assay (DjinniChip) for diagnosis of ocular C. trachomatis for use by trachoma control programmes. METHODS: The study was conducted in the UK, Germany and Tanzania. For clinical testing in Tanzania, specimens from a sample of 350 children between the ages of 7 to 15 years, which were part of a longitudinal cohort that began in February 2012 were selected. Two ocular swabs were taken from the right eye. The second swab was collected dry, kept cool in the field and archived at - 80 °C before sample lysis for DjinniChip detection and parallel nucleic acid purification and detection/quantification by qPCR assay. RESULTS: DjinniChip was able to reliably detect > 10 copies of C. trachomatis per test and correctly identified 7/10 Quality Control for Molecular Diagnostics C. trachomatis panel samples, failing to detect 3 positive samples with genome equivalent amounts ≤ 10 copies. DjinniChip performed well across a range of typical trachoma field conditions and when used by lay personnel using a series of mock samples. In the laboratory in Tanzania, using clinical samples the sensitivity and specificity of DjinniChip for C. trachomatis was 66% (95% CI 51-78) and 94.8 (95% CI 91-97%) with an overall accuracy of 90.1 (95% CI 86.4-93). CONCLUSIONS: DjinniChip performance is extremely promising, particularly its ability to detect low concentrations of C. trachomatis and its usability in field conditions. The DjinniChip requires further development to reduce inhibition and advance toward a closed system. DjinniChip results did not vary between local laboratory results and typical trachoma field settings, illustrating its potential for use in low-resource areas to prevent unnecessary rounds of MDA and to monitor for C. trachomatis recrudescence.